A number of pathways and newly identified genes appear to be critical for the differentiation and identification of autoreactive Thl T cells. Specifically, T-bet is the master regulator of Thl T cells and TIMS defines Thl T cells in murine systems; in contrast, GATA-3 is a regulator of Th2 differentiation. We have previously cloned T cells from the CSF of patients with MS and demonstrated a significantly higher frequency of IFNy secreting T cells in the CSF of patients with MS as compared to T cells isolated from the CSF of subjects without inflammation. Recent data from Kuchroo (Project 2) demonstrated that engagement of TIMS with TIMS ligand regulated both the function of Thl cells and the ability to induce tolerance. Moreover, TIMS deficient mice were refractory to the induction of high dose tolerance in EAE. We will examine the hypothesis that loss of TIMS expression on T cells in part underlies the loss of tolerance to self- antigens in autoimmune disease and is involved in the pathophysiology of MS. Specifically, in aim 1 we will examine the function of TIMS expression on human T cells, determining the regulation and stability of TIMS expression on differentiated human Thl and Th2 cells from healthy subjects by investigating the relationship between TIMS and T-bet expression with anergy. This aim will directly translate the findings by Glimcher (Project 1) and Kuchroo (Project 2) and will allow us in our second aim to directly examine T cell function in patients with MS, where we will determine whether altered expression of TIMS functionally characterizes the loss of tolerance by T cells from the CNS of patients with MS. This aim is based on new preliminary data suggesting that the high IFNv secreting CD4 T cells in the CSF of patients with MS, but not CD4 T cells from control subjects, exhibit altered levels of TIMS. In the last aim, we will directly interrogate the CNS of patients with MS and ask if there a loss of TIMS expression associated with high IFNy expression of CD4 T cells in the plaque tissue of patients with MS. This aim addresses the hypothesis that loss of TIMS on T cells infiltrating plaque tissue of chronic MS lesions underlies the loss of tolerance and protracted expansion of autoreactive T cells in the brain. These experiments will directly translate the basic immunologic discoveries and findings in the EAE model in attempts to understandthe mechanism for loss of T cell tolerance to myelin antigens in patients with multiple sclerosis.